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First published online 5 November 2003
doi: 10.1242/dev.00844

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Na,K-ATPase is essential for embryonic heart development in the zebrafish

¹ Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA
² Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA
³ Jonsson Cancer Center, University of California, Los Angeles, Los Angeles, CA 90095, USA
⁴ Cardiovascular Research Laboratory, University of California, Los Angeles, Los Angeles, CA 90095, USA
⁵ Department of Pediatrics, University of California, Los Angeles, Los Angeles, CA 90095, USA
⁶ Developmental Biology Laboratory, Massachusetts General Hospital, Charlestown, MA 02129, USA
⁷ Institute of Molecular and Cell Biology, College of Life Science, National Taiwan University, Taipei, Taiwan

View larger version (118K): [in a new window]   Fig. 1. Brain and body axis defects in had mutants. (A,B) Live wild-type (left) and had mutant (right) embryo. The body axis is normal in had mutants at 24 hpf (A) but becomes curly by 48 hpf (B). (C,F) By 24 hpf, the boundary of midbrain and hindbrain is distinctive in wild-type embryos (C, arrow), but not had mutants (F, arrow). (D,G) Twoday-old wild-type (D) and had mutant embryos (G). Note that twoday-old had mutant has developed edema. (E,H) pax2 expression pattern in wild-type (E) and had mutant (H) embryos at 24 hpf.

View larger version (77K): [in a new window]   Fig. 2. Primitive heart tube extension is perturbed in had mutant embryos. (A-F) All panels show dorsal views, anterior to the bottom. Cardiac cells (arrows) are visualized by in situ hybridization analysis using a vmhc probe. (A) Bilateral cardiac primordia have fused at the midline in wild type zebrafish embryos at 22 hpf. (B) By 24 hpf, the fused heart has grown into a tubular structure, known as the primitive heart tube. (C) By 28 hpf, a long tubular heart is clearly visible in the wild types. In had mutant embryos, the fusion of bilateral cardiac primordia appears normal (D), but primitive heart tube extension is severely defective (E,F). (E) The primitive heart of the had mutant is a cone-shaped structure at the midline at 24 hpf, and the primitive heart tube is significantly shorter at 28 hpf (F), compared with that observed in wild-type siblings (C).

View larger version (87K): [in a new window]   Fig. 3. The cmlc2-driven EGFP is expressed in cardiomyocytes. (A) The wild-type primitive heart is a long tubular structure. (B) The primitive heart tube is short in the had mutant embryo, but has a stronger EGFP signal. Arrows point to the heart. (C) A typical image of dissociated cardiomyocytes from a cmlc2:EGFP embryo at 24 hpf. (D) Comparison of the numbers of cardiomyocytes in wild-type and had mutant embryos at 24 hpf. The y-axis shows the number of EGFP-positive cells.

View larger version (75K): [in a new window]   Fig. 4. Cardiomyocyte differentiation defects in had mutants. (A,B) Transverse sections of two-day-old wild-type (A) and had mutant (B) heart. Arrow indicates myocardium; arrowhead, endocardium. (C-H) Ventral views of 50 hpf embryos, head to the top, solid line marks the site of the constriction between the ventricle (v) and the atrium (a). Cardiac expression of vmhc (C,D), irx1 (E,F) and versican (G,H) was detected by in situ hybridization. The hearts of had mutants are smaller and dysmorphic (B,D,F,H) compared with those in wild types (A,C,E,G). By 50 hpf, expression of vmhc is restricted to ventricles in wild types (C), but expression of this gene extends to the atrium in had mutants (D). Cardiac expression of irx1 is restricted to ventricles in wild-type embryos (E), but is severely reduced in the heart of had mutants (F, arrow). versican is expressed at the boundary of the atrium and the ventricle in wild-type embryos (G), but is expressed throughout had mutant hearts (H).

View larger version (44K): [in a new window]   Fig. 5. had encodes the zebrafish Na,K ATPase 1B1 isoform. (A) Linkage analysis shows 98 recombinants to z5508, 41 recombinants to z3705 and no recombinants to Z6384 of LG1 in 848 meiosis. (B) Schematic cDNA and genomic structures of zebrafish Na,KATPase 1B1. Dark boxes depict transmembrane domains (TM1-TM10). Arrows indicate positions of primers (5F and 5R) used for PCR analysis. Four exons (depicted by blue-boxes, labeled as exon A-D) were amplified from the genomic DNA using the 5F and 5R primers. A deletion at the 3' boundary of exon C (red bar) was detected in had mutants. (C) Genomic sequence of 1B1 between the 5F and 5R primer sequences. The 2 bp-insertion is shown in blue and sequences deleted in had are shown in red. (D) RT-PCR analysis shows two alternative splicing variants (arrows) in had mutants. (E) Predicted structure of the Na,K-ATPase 1B1 protein. Amino acids encoded by exon B are shown in green and those encoded by exon C are in yellow. The purple region represents sequences between TM7 and TM8, which include crucial elements for the interaction of the and ß subunits. (F) The 1B1 isoform is highly expressed in the two-day old wild-type embryonic heart, but is not detectable in had mutant heart. Arrows point to the heart.

View larger version (86K): [in a new window]   Fig. 6. Functional analyses of cardiac Na,K-ATPase isoforms. (A-E) Lateral views of 50 hpf embryos, head to the top. Ouabain treatment (C) and 1B1MO injection (D) create embryos morphologically indistinguishable from had mutants (B). The gross morphology of 2MO-injected embryos (E) is similar to that of wild-type controls (A). (F-O) Cardiac tissues are visualized by in situ hybridization using a vmhc probe. (F-J) Dorsal views of embryos at 24 hpf, head to the bottom; the arrows point to the heart. (K-O) Ventral views of 50 hpf embryos, head to the top; the solid line marks the constriction between the atrium (a) and the ventricle (v). (F) Wild-type control embryos at 24 hpf. Ouabain treatment (H) and 1B1MO (I) injection perturbed primitive heart tube extension in oneday-old embryos similar to the had mutation (G). (K) vmhc expression is restricted to the ventricle of wild-type control embryos at 50 hpf. The expression of vmhc extends into the atrium of had (L), ouabain-treated (M) and 1B1MO-injected (N) embryos. (J) Cardiac jogging is affected by 2MO. Some 2MO-injected embryos have the primitive heart tube placed on the right side of the embryos. After two days of development, abnormal cardiac looping is observed in 2MO-injected embryos. Some fail to loop and some have the ventricles on the left of the atrium (O).

View larger version (118K): [in a new window]   Fig. 7. Rescue of had cardiac phenotypes. At 24 hpf, the heart is a tubular structure in the wild-type embryos (A), but remains as a shallow cone in had mutant embryos (B). (C) Injecting 1B1 mRNA into had mutant embryos results in the development of a normal tubular heart. (D) Hearts of had embryos injected with 2 mRNA remain as a shallow cone.